Membrane fusion at neuronal synapses is a highly regulated process that is triggered by micromolar concentrations of Ca 2+ in the presynaptic cell. Although assembly of the SNARE complex is necessary for fusion, the SNARE proteins themselves are not directly regulated by Ca 2+. Synaptotagmin I is a vesicle associated membrane protein that is believed to function as the Ca 2+ sensor in neuronal exocytosis. It is anchored to the vesicle membrane by a single transmembrane segment at its N-terminus and contains two C2 domains that have been shown to bind membranes in a Ca2+-dependent fashion. Synaptotagmin is also reported to bind SNAREs. At the present time, the nature of the synaptotagmin interactions that mediate fusion are not understood. It is not known whether the C2 domains in synaptotagmin trigger fusion by binding to membranes alone, or whether they also interact with and modulate components of the SNARE complex. The proposed work will utilize magnetic resonance methods, such as site-directed spin labeling, to determine the membrane and protein interactions made by synaptotagmin. These experiments will determine whether synaptotagmin's two C2 domains assume cis or trans configurations when presented with target membranes, and determine whether these domains interact with SNAREs in reconstituted systems. Proposed experiments will test the effect of point mutations on the membrane bound orientation of synaptotagmin as well as hypothesized mechanims for synaptotagmin action. Finally, both the strength and stoichiometry of PI(4,5)P2 interactions with synaptotagmin and SNAREs will be characterized. Understanding the mechanism of Ca2+-triggered fusion will have direct consequences in neurobiology for understanding synaptic transmission and potentiation. In general, understanding how fusion is triggered, and the role played by PI(4,5)P2, may have consequences for understanding other fusion related processes, such as intracellular transport, host defense (killing of microorganisms, immune response), and human physiology and disease (e.g., glucose regulation/diabetes, allergic response).